There is no doubt that N-nitrosamines, as one important class of potential human carcinogens, require metabolic activation to initiate the oncogenic process. Although various metabolic pathways are available to these compounds, it appears that enzymic alpha-hydroxylation eventually affords the ultimate carcinogenic agent (carbenium ion). It has been assumed that this enzymic activation occurs in the cytoplasm, and therefore it follows that the alpha-hydroxynitrosamine formed is sufficiently stable to diffuse through the cytoplasm, penetrate the nuclear envelope and then decompose to the "ultimate" carcinogenic form that covalently binds to nuclear DNA. We propose to determine if in fact alpha-hydroxynitrosamines can penetrate a nuclear membrane by incubating isolated intact liver nuclei with (14-C)-alpha-acetoxynitrosamines and hog liver esterase and quantitating the levels of radioactive binding to nuclear DNA. Concurrent with this work on a "transportable" metabolite, we plan to determine if the activation of nitrosamines into reactive electrophilic species can be mediated by nuclear enzymes. This will be accomplished by the incubation of (14-C)-labeled nitrosamines, that induce liver cancer in rats, with purified intact liver nuclei from Wistar rats and assaying for bound (14-C)-label in the nuclear DNA, RNA and protein. The effect of pretreatment with the respective nitrosamines on binding by nuclei shall be determined. Also the identification of sites of P-450 activity in the nuclear membrane will be elucidated by electron microscopy cytochemical studies. To put the results of the binding studies with isolated nuclei in perspective we plan to evaluate the extent of binding to rat liver macromolecules upon in vitro incubation of (14-C)-nitrosamines with isolated whole hepatocytes. Overall, these investigations should add to current knowledge concerning the activation of nitrosamines, in terms of how and where this activation occurs and in the development of model systems which will facilitate the studying of DNA modification by chemicals, a process thought to be the first step in nitrosamine chemical carcinogenesis.